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Lecture 11. Test next week in class Protein structure. Collagen. Most abundant protein of vertebrates. Extracellular protein-insoluble fibers, great tensile strength Major component of connective tissues (bone, teeth, cartilage, tendon, ligament, etc.)

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Lecture 11
Lecture 11

  • Test next week in class

  • Protein structure


Collagen
Collagen

  • Most abundant protein of vertebrates.

  • Extracellular protein-insoluble fibers, great tensile strength

  • Major component of connective tissues (bone, teeth, cartilage, tendon, ligament, etc.)

  • Type I collagen-3 polypeptide chains, 285 kD.

    • 3000 Å long and 14 Å diameter.

  • Distinct amino acid composition; 1/3 are Gly and 15-30% are Pro and 4-hydroxyprolyl (Hyp) residues.


Collagen1
Collagen

  • Collagen has a triple-helical structure

  • Amino acid sequence has repeating triplets of Gly-X-Y with X=Pro and Y= Hyp over 1011 residues out of 1042 residue polypeptide.

  • Forms a right-handed triple helical structure.


The triple helix of collagen
The triple helix of collagen.

  • Shows how left-handed polypeptide helices are twisted together to form a right-handed superhelical structure.

  • Individual polypeptide has 3.3 residues per turn and pitch of 10 Å.

  • The collagen triple helix has 10 Gly-X-Y units per turn and a pitch of 86.1 Å.

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Lecture 11

Figure 8-30b X-Ray structure of the triple helical collagen model peptide (Pro-Hyp-Gly)10 in which the fifth Gly is replaced by Ala. (b) View along helix axis.

Page 235


Figure 8 31 electron micrograph of collagen fibrils from skin
Figure 8-31 Electron micrograph of collagen fibrils from skin.

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Figure 8 32 banded appearance of collagen fibrils
Figure 8-32 Banded appearance of collagen fibrils.

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Lecture 11

Figure 8-30c X-Ray structure of the triple helical collagen model peptide (Pro-Hyp-Gly)10 in which the fifth Gly is replaced by Ala. (c) A schematic diagram.

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Figure 8 33 a biosynthetic pathway for cross linking lys hyl and his side chains in collagen
Figure 8-33 A biosynthetic pathway for cross-linking Lys, Hyl, and His side chains in collagen.

  • Collagen fibrils are covalently cross-linked.

  • Collagen almost no cysteine.

  • It is cross linked y Lys and His.

  • Lysyl oxidase coverts lysine to allysine.

  • Allysine are condesed to allysine aldol.

  • This reacts with His to form Aldol-His.

  • Aldol-His reacts with 5-hydroxy-Lys crosss linking the four side chains.

Page 238


Table 8 3 the arrangement of collagen fibrils in various tissues
Table 8-3 The Arrangement of Collagen Fibrils in Various Tissues.

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Globular proteins
Globular proteins

  • Diverse group of proteins that exist as compact spherical molecules.

  • Enzymes, transport, and receptor proteins.

  • Most structural information from X-ray crystal structure and NMR.

  • X-ray crystallography directly images molecules.

  • X-ray wavelengths are small 1.5 Å (visible light is 4000 Å)

  • X-rays generated by synchrotrons, a type of particle accelerator to make X-rays of high intensity.


Crystalline proteins
Crystalline proteins

  • Molecules in protein crystals are arranged in regularly repeating 3-D lattices.

  • Unlike other small organic or inorganic molecules, proteins are highly hydrated (40-60% H2O)

  • Water is required for the native structure of the proteins.

  • Generally disordered by >1 Å.

  • Typical resolution is 1.5 to 3.0 Å.


Figure 8 36a electron density maps of proteins
Figure 8-36a Electron density maps of proteins.

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Figure 8 36b electron density maps of proteins
Figure 8-36b Electron density maps of proteins.

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Figure 8 36c electron density maps of proteins
Figure 8-36c Electron density maps of proteins.

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Lecture 11
Figure 8-37 Sections through the electron density map of diketopiperazine calculated at the indicated resolution levels.

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Crystalline proteins1
Crystalline proteins

  • Crystalline proteins assume the same structure they have in solution

  • Crystals have 40-60% water content (similar to most cells)

  • Proteins may crystallize in of several forms depending on conditions. Different crystal forms of the same protein have identical conformations.

  • Many enzymes are catalytically active in the crystalline state.


Nmr for protein structure determination
NMR for protein structure determination

  • Use of 2D NMR

  • Yields interaatomic distances between specific protons that are <5 Å apart.

  • Interproton distances through space can be determined by nuclear Overhauser effect spectroscopy(NOESY)

  • Interproton distance through bonds as determined by correlated spectroscopy(COSY).

  • Present methods are good only with molecular masses up to 40 kD.

  • Usually well correlated with X-ray data, but sometimes differs.

  • NMR can probe motions over time scales of 10 orders of magnitude so can be used to study protein folding and dynamics.


Lecture 11

Figure 8-38a The 2D proton NMR structures of proteins.(a) A NOESY spectrum of a protein presented as a contour plot with two frequency axes w1 and w2.

Off diagonal peaks (cross peaks) occur from interaction of 2 protons that are <5 Å apart in space and whose 1D-NMR peaks are located where the horizontal and vertical lines cross through the cross peak intersect the diagonal.

Nuclear Overhauser Effect (NOE)

c

b

b

b

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c

c

a

a

d

d

a

d


Lecture 11

Figure 8-38b The 2D proton NMR structures of proteins.(b) The NMR structure of a 64-residue polypeptide comprising the Src protein SH3 domain.

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Tertiary structure
Tertiary structure

  • Tertiary structure is the three dimensional arrangement of a protein.

  • Includes the folding of secondary structural elements and spatial dispositions of the side chains.

  • Determined by X-ray crystallography and NMR


Lecture 11

Figure 8-39a Representations of the X-ray structure of sperm whale myoglobin. (a) The protein and its bound heme are drawn in stick form.

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Lecture 11

Figure 8-39b Representations of the X-ray structure of sperm whale myoglobin. (b)A diagram in which the protein is represented by its computer-generated Ca backbone.

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Lecture 11

Figure 8-39c Representations of the X-ray structure of sperm whale myoglobin. (c)A computer-generated cartoon drawing in an orientation similar to that of Part b.

Page 244


Globular proteins have both helices and sheets
Globular proteins have both  helices and  sheets

  • Most proteins have a significant amount of secondary structure

  • On average 31%  helix, 28%  sheet, and a total content of helices, sheets, turns and  loops comprising 90% of the structure of a protein.


Figure 8 40 the x ray structure of jack bean protein concanavalin a
Figure 8-40 The X-ray structure of jack bean protein concanavalin A.

Page 245


Figure 8 41 human carbonic anhydrase
Figure 8-41 Human carbonic anhydrase.

Page 245


Side chain location varies with polarity
Side chain location varies with polarity

  • Globular proteins lack the repeating sequences responsiblee for the regular conformations of fibrous proteins.

  • The amino acid side chains in globular proteins are distributed according to polarities.

  • Nonpolar residues (Val, Leu, Ile, Met, and Phe) occur in the interior of a protein.

  • Charged polar residues (Arg, Lys, His, Asp, Glu) are mostly located on the surface of a protein.

  • Uncharged polar residues (Ser, Thr, Asn, Gln, Tyr, and Trp) are usually on the surface but can occur in the interior of the protein.

    • If in the interior, they are H-bonded to neutralize their polarity.


Figure 8 42a the x ray structure of horse heart cytochrome
Figure 8-42a The X-Ray structure of horse heart cytochrome.

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Lecture 11

Figure 8-43a The H helix of sperm whale myoglobin. (a)A helical wheel representation in which the side chain positions about the a helix are projected down the helix axis onto a plane.20

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Figure 8 43b the h helix of sperm whale myoglobin b a skeletal model viewed as in part a
Figure 8-43b The H helix of sperm whale myoglobin.(b) A skeletal model, viewed as in Part a.

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Lecture 11

Figure 8-43c The H helix of sperm whale myoglobin.(c) A space-filling model, viewed from the bottom of the page in Parts a and b and colored as in Part b.

Page 247


Figure 8 44 a space filling model of an antiparallel b sheet from concanavalin a
Figure 8-44 A space-filling model of an antiparallel b sheet from concanavalin A.

Page 247


Lecture 11

Energy diagram of the protein folding process.

The completely unfolded protein is thought to be in the least stable form.

For most proteins, the native conformation is the most thermodynamically stable and the only form that is biologically active.


Lecture 11

Denaturation and renaturation of a protein

  • The complete loss of organized structure in a protein is called “denaturation”.

  • Denaturation results in loss of biological activity!

  • Denaturation process occurs during cooking an egg.

  • Denaturants include:

  • Large evil fire ants??

  • Heat

  • Organic solvents

  • Urea

  • Detergents

  • Acid or base

  • Shear stress

  • Hydrophobic interfaces


Lecture 11

Structural Motifs in Proteins.

  • Individual units of 2ndary structure combine into stable, geometrical arrangements.

  • Called supersecondary structure or motifs.

  • Are often repeated in same protein, different proteins.

  • Certain motifs have associated biological functions:

    a-Helix-loop-a-helix motif binds DNA, sequesters calcium ion.

  • Secondary structures often depicted as ribbon diagrams

  • Ribbons invented by Jane Richardson, originally drawn by hand, now done by computer programs.


Lecture 11

Some common structural motifs of folded proteins

The aa motif

(helix-turn helix)



Lecture 11

Some common structural motifs of folded proteins

c) The bbbb “Greek Key” motif



Lecture 11

Several bab motifs combine to form a superbarrel in the glycolysis enzyme triose phosphate isomerase (TIM barrel)


Quaternary structure
Quaternary structure

  • Spatial arrangement of protein subunits.

  • Polypeptide subunits associate in a geometrically specific manner.

  • Why subunits?

  • Easier to repair self-assembling single subunit vs. a large polypeptide.

  • Increasing a protein’s size through subunits is more efficient for specifying the active site.

  • Provides a structural basis for regulating activity.


Lecture 11

Domains in proteins.

  • Common sequence regions in native proteins can fold up to form compact structures called “domains”.

  • Domains can range in size from 50-400 amino acids, have upper limit in forming compact hydrophobic core.

  • Domains are a type of folding motif, typically have separate hydrophobic core.

  • Larger proteins are composed of multiple domains, often connected by flexible linker peptide regions.

  • Classic example: antibodies


Antibody immunoglobulin domains

Antibody Immunoglobulin Domains

Structural elements of IgGs:

Naturally occurring immunoglobulins (IgG molecules) have identical heavy chains and light chains giving rise to multiple binding sites with identical specificities for antigen.


Antibody immunoglobulin domains1

Antibody Immunoglobulin Domains

Antibodies are composed of:

V (for variable) regions - encodes the antigen binding activity

C (for constant) regions - encodes immune response signal/effector functions:

Complement fixation (activation of complement cascade)

Binding and activation of Ig receptors (transport from maternal source, activate immune system T cells to engulf, destroy foreign cells, particles, proteins)

Also binds bacterial Protein A, Protein G (used in purification)

Note: dashed lines indicate

interchain disulfide bonds


Antibody immunoglobulin domains2

Antibody Immunoglobulin Domains

There is a conserved glycosylation site in the CH2 domain of IgG (purple region).

A carbohydrate is covalently attached here by postranslational modification.


Antibody immunoglobulin domains3

Antibody Immunoglobulin Domains

IgG secondary/tertiary structure: multiple beta-sheet domains.

Termed “immunoglobulin domain”.

Repeated motif in many immune and receptor proteins.


Antibody immunoglobulin domains4

Antibody Immunoglobulin Domains

Modes of Flexibility of IgG structure


Subunit interactions
Subunit interactions

  • Identical subunits in a protein are called protomers

  • Proteins with identical subunits are oligomers.

  • Hemoglobin is a dimer (oligomer of two protomers) of protomers.